Supercritical CO2 extraction of oil, fatty acids and ...

Food and Chemical Toxicology 83 (2015) 275e282

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Supercritical CO2 extraction of oil, fatty acids and flavonolignans from milk thistle seeds: Evaluation of their antioxidant and cytotoxic activities in Caco-2 cells Naila Ben Rahal a, *, Francisco J. Barba b, Danielle Barth a, Isabelle Chevalot a a b

Laboratoire R eactions et G enie des Proc ed es UMR CNRS 7274, Universit e de Lorraine, 1, rue Grandville BP20451, 54001 Nancy, France Nutrition and Food Science Area, Faculty of Pharmacy, Universitat de Val encia, Avda. Vicent Andr es Estell es, s/n., 46100 Burjassot, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2015 Received in revised form 22 June 2015 Accepted 8 July 2015 Available online 11 July 2015

The optimal conditions of supercritical carbon dioxide (SC-CO2) (160e220 bars, 40e80 C) technology combined with co-solvent (ethanol), to recover oil, flavonolignans (silychristin, silydianin and silybinin) and fatty acids from milk thistle seeds, to be used as food additives and/or nutraceuticals, were studied. Moreover, the antioxidant and cytotoxic activities of the SC-CO2 oil seeds extracts were evaluated in Caco-2 carcinoma cells. Pressure and temperature had a significant effect on oil and flavonolignans recovery, although there was not observed a clear trend. SC-CO2 with co-solvent extraction at 220 bars, 40 C was the optimum treatment to recover oil (30.8%) and flavonolignans from milk thistle seeds. Moreover, linoleic (47.64e66.70%), and oleic (19.68e24.83%) acids were the predominant fatty acids in the oil extracts recovered from milk thistle under SC-CO2. In addition, SC-CO2 extract showed a high antioxidant activity determined by DPPH and ABTS tests. Cytotoxic activities of silychristin, silydianin and silybinin and the obtained SC-CO2 extract (220 bars, 40 C) were evaluated against Caco-2 cells. The SC-CO2 extract inhibited the proliferation of Caco-2 cells in a dose-responsive manner and induced the highest percentage of mortality of Caco-2 cells (from 43 to 71% for concentrations from 10 up to 100 mg/ ml of SC-CO2 oil seeds) © 2015 Elsevier Ltd. All rights reserved.

Keywords: Milk thistle seeds Oil Supercritical CO2 Fatty acids Flavonolignans Caco-2 carcinoma cells

1. Introduction In the last two decades, the increasing ban on the use of artificial food additives as well as the growing interest in use of active compounds from natural sources that can contribute to promote consumer's health and prevent diseases, has led to both food industry and food researchers to explore natural sources of food additives and/or nutraceuticals (Bearth et al., 2014; Downham and Collins, 2000; Parniakov et al., 2014; Thurmond, 2014). For instance, Silybum marianum (family: Asteracae), commonly known as milk thistle, is a native plant from the Mediterranean area, which can constitute an important industrial agricultural crop. Milk thistle leaves and flowers have been used as a vegetable for salads and a substitute for spinach. On the other hand, S. marianum seeds are roasted for use as a coffee substitute. Moreover, the oil extracted from milk thistle seeds can be used

* Corresponding author. E-mail address: [email protected] (N. Ben Rahal). http://dx.doi.org/10.1016/j.fct.2015.07.006 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

in the anticholesterol diets for cardiovascular disease prevention (El-Mallah et al., 2003) as it constitutes a good source of unsaturated fatty acids (56% polyunsaturated and 21% monounsaturated) (Yin et al., 1998), vitamin E (50e60 mg/100 g) (Hadolin et al., 2001), phenolic compounds and flavonoids (0.25%) (Li et al., 2012; Pereira et al., 2015; Wallace et al., 2003). In addition, milk thistle seeds contain active compounds such as silymarin. Silymarin has been known since centuries and recommended in traditional European and Asian medicine, mainly for treatment of liver disorders (Hadolin et al., 2001). These seeds contain silymarin complex, which consists of four flavonolignans (Pereira et al., 2015; Wallace et al., 2003): silychristin (SCN), silydianin (SDN), silibinin (SBN) and taxifolin (TXF) Fig. 1. The anticancer activity of silymarin as well as silibinin was demonstrated against various cancer cells such as breast, skin, colon, cervix, ovary, prostate, lung and hepatocellular cancers (BoschBarrera and Menendez, 2015; Chu et al., 2004; Eo et al., 2015; Fan et al., 2014; Jiang et al., 2015; Mastron et al., 2015; Sharma et al., 2003; Thelen et al., 2004; Tyagi et al., 2004; Varghese et al., 2005). Therefore, the recovery of these active compounds from milk

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Fig. 1. Flavonolignans structure (SBN: Silybinin, TXF: Taxifolin, SCN: Silychristin, SDN: Silydianin) (Quaglia et al., 1999).

thistle seeds constitutes an important challenge. Traditionally, conventional solvent extraction has been used for these purposes (Hadolin et al., 2001). However, conventional techniques involve the use of toxic solvents (i.e. hexane) and long extraction times. Thus, at this stage of development, there is a need to establish new processes in full correspondence with green extraction concept, which can reduce solvent consumption, extraction time and toxicity (Chemat et al., 2012; Rosello-Soto et al., 2015). In this way, supercritical fluid extraction (SFE) technique is a promising alternative to conventional extraction methods (Rawson et al., 2012). Carbon dioxide in its supercritical form (SCeCO2) has been widely used in many SFE applications and, it has gained increased attention in the food, pharmaceutical and cosmetic industries to obtain high-added value compounds such as flavors, fragrance ingredients, food additives and/or nutraceuticals. Some of the main advantages of using CO2 as supercritical fluid are the low toxicity, and non-combustibility of this compound. Moreover, CO2 presents an adjustable selectivity towards the different components, and does not react with targeted compounds (Friedrich and Pryde, 1984). Depending on pressure and temperature, liquid CO2 can be adjusted to be more liquid-like or gas-like (Barba et al., 2014; Deng et al., 2014; Reverchon and Senatore, 1992). Moreover, the use of co-solvent gives an additional advantage to SFE process as the solubility increase caused by co-solvent allows pressure extraction decrease. This selectivity is due to specific interactions with the solute (Senorans et al., 2000). To the best of our knowledge, there is a lack of information about the effects of SFE with co-solvent on the recovery of fatty acids, and flavonolignans from milk thistle seeds (Celik and Guru, 2015), as well as the potential cytotoxicity of these compounds on cancer cell lines. Thus, the aims of the present work are: 1) to evaluate the effects of different SC-CO2 with co-solvent conditions to recover oil, fatty acids and flavonolignans from S. marianum seeds. 2) To study the antioxidant (DPPH and ABTS tests) and cytotoxic activities of S. marianum oil seeds in Caco-2 carcinoma cells. 2. Materials and methods 2.1. Samples Milk thistle samples were collected from the north of Tunisia. Sheets, stems, roots and impurities were eliminated thanks to a traditional sieve. Seeds were crushed to obtain a fine powder and then stored at 4 C until needed for analysis. Seed powders were

separated into two sections according to their particle size (310 mm and 620 mm of particle diameter). The values of dry matter (%) and mineral content (%) of seed powders were 95.95 ± 0.36 and 4.81 ± 0.06, respectively. 2.2. SC-CO2 extraction The experiments were carried out in a dynamic extraction unit as it is shown in Fig. 2. The experimental design was previously described (Bensebia et al., 2009). Thirty g of dried powder seeds (particle diameter ¼ 310 mm) were packed into a sample unit. In order to prevent the entrainment of milk thistle during extraction process, and to improve the gas homogenization in the SC-CO2 equipment, glass wool was placed at the top and bottom of sample unit. The sample was then allowed to reach the constant extraction temperature before charging CO2 into the high-pressure pump from the storage cylinder. Ethanol (co-solvent) was added to increase polyphenols solubility. In fact, SC-CO2 allows to solubilize nonpolar compounds (low molecular weight) in low critical temperature (Tc ¼ 31 C), thus facilitating the extraction of sensitive products at low temperature. Ethanol was introduced with a Gilson pump with a flow rate of 5 ml/min for 15 min during contact time of the plant and SC-CO2 (before pumping the supercritical CO2). The CO2 gas was further compressed to the desired pressure of the pump. Static and dynamic times were 30 and 120 min (¼time to exhaustion of oil in the seed), respectively. Samples were withdrawn every 15 min. The separation of CO2 extract was carried out at a temperature of 50 C. Co-solvent (ethanol) was removed using a rotary evaporator. Finally, oil samples were kept in a freezer, until needed for analysis. The operating conditions such as pressure, temperature, density of solid and fluid, and solubility were fixed as it is shown in Table 1. 2.3. Gas chromatographyemass spectroscopy (GCeMS) analysis The GCeMS analysis was carried out on an Hewlett Packard 5890 II gas chromatograph with a HP-5MS 5% phenylmethylsiloxane capillary column (30 m 0.25 mm i.d., film thickness 0.25 mm). Helium was used as a carrier gas at a flow rate of 20 mL/min. Each sample (1 mL) was injected into the column at a split ratio of 15:1. The mass spectrometer was operated in electronimpactionization (EI) mode by the energy of 70eV. The scanning range was 50e600 amu and the scanning rate was 0.5s/scanning. The individual identification of compounds was based on matching


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Fig. 2. Dynamic extraction apparatus (Goodarznia and Eikani, 1998).

Table 1 Molar volume of CO2 (MvCO2 ) and CO2 density (rCO2) under supercritical carbon dioxide (SCeCO2) (160e220 bars, 40e80 C) conditions. Pressure (bars)

Temperature ( C)

MvCO2 (cm3/mol)

rCO2 (kg/m3)

160

40 60 80 40 60 80 40 60 80

60.43 77.48 103.17 57.83 70.96 90.37 54.17 63.27 75.83

730 570 430 760 620 490 810 700 580

180

220

their recorded mass spectra with those of NBS75K library data provided by the software of GCeMS system (NIST, 1986). 2.4. High performance liquid chromatography (HPLC) analysis HPLC method is adapted from Quaglia et al. (1999) method. A Shimadzu LC-10AT VP chromatograph equipped with a Varian photodiode array detector, was used for the analyses. The chromatograph was controlled and the data evaluated by a computer Flyer Pentium, interface D 7000. Sample solutions were injected using a 20 ml sample loop. The flavonolignans separation was carried out using a stationary phase: C18 pre-column (Alltech) and C18 column (150 mm 4.6 mm, 5 mm) (Varian XRs) maintained at 40 C. The mobile phases used were described by Quaglia et al. (1999). The elution was made at a flow rate of 1 ml/min in these gradient conditions: Solvent A: Water, acidified with 10% H3PO4 (pH 2.6); Solvent B: acetonitril; Solvent C: methanol (flow rate of 1 ml/min). At t ¼ 0 min: 63% of solvent A, 15% of solvent B, 22% of solvent C. At 7.5 min: 63% of solvent A, 15% of solvent B, 22% of solvent C. At 8.5 min: 40% of solvent A, 20% of solvent B, 40% of solvent C. At 15 min: 40% of solvent A, 20% of solvent B, 40% of solvent C. The photodiode array detector conditions were: l ¼ 330 nm. Acquisition rate of spectra 1600 ms. Spectral bandwidth for each channel 4 nm. Wavelength range: 220e350 nm. The standard solutions were prepared from three flavonolignans (SBN 97.1%, SCN 82.2%, SDN 93.2%) (ChromaDex, France).

2.5. Antioxidant tests 2.5.1. DPPH test The evaluation of the antioxidant activity of silymarin was performed with a DPPH radical and measured according to the method described by Brand-Williams et al. (1995). The assays were performed in 2 ml reaction mixtures containing 1.95 ml of 0.1 mM DPPH-ethanol solution and 0.05 ml of the samples. The inhibitory effect of the different concentrations of silymarin extracts (0.05 102 mg/ml) on DPPH were measured by spectrophotometeric method. The absorbance of the reaction mixtures at 517 nm was continuously monitored for 90 min. 2.5.2. ABTS test The standard TEAC (Trolox Equivalent Antioxidant Capacity) assay described by Re et al. (1999) was used with minor modifications for the determination of TEAC value. The absorbance of the mix solution (ABTS $þ radical þ phosphate buffer (pH 7.4)) was measured at 734 nm (AABTS). One hundred microlitres of the diluted sample were added in a tube, this solution was mixed quickly, and the absorbance at 734 nm was measured after 60s. The decrease in absorbance (DA ¼ AABTS Asilymarin) after 60 s was calculated for each diluted silymarin sample. The decrease in absorbance caused by the addition of Trolox as the standard was measured by the same procedure for each concentration of Trolox (50e400 mol/L) and the calibration curve for the decrease in absorbance (DA ¼ AABTS ATrolox) of Trolox vs. Trolox concentration was constructed by linear regression. ABTSþ radical inhibition percentage caused by each diluted sample was plotted against the non-diluted sample volume in mixture reaction. 2.6. Cytotoxic activity Caco-2 cells (colon cancer cell line) were cultivated in Dulbecco's modified eagle medium (DMEM) with high glucose (4.5 g/l) (Sigma, Germany), and supplemented with 10% fetal calf serum (FCS) (EuroBio, France), 2 mM L-glutamine, and 1% nonessential amino acids (GIBCO, USA). The cells were usually splitted when reaching confluence (5e7 days). They were first rinsed with Dulbecco's phosphate-buffered saline without calcium (D-PBS) (Sigma, Germany) and then trypsinised with a solution containing 0.25%

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trypsin and 1 mM EDTA (GIBCO, USA). Caco-2 cells were feeded into 96-well microplates at 1 104 cells/well in 200 ml of appropriate culture medium. The action of trypsin was stopped by the addition of culture medium containing fetal calf serum (10%) and the cells were left in solution. Subsequently, the cells were recovered by centrifugation at 800 rpm/min for 5 min. Then, the cultures were maintained in an incubator at 37 C under a moisture saturated atmosphere containing 5% CO2. In order to ensure the nutrient supply to the cells and removing cellular metabolites capable of inhibiting cell growth, a change of culture medium was needed every 48 h. Cell growth was evaluated daily using a microscope in opposite phase. The cells were grown to 80% confluence where a concentration of 0.6 105 cells/cm was reached. After 24 h, the cells were exposed to various concentrations of the compounds solubilized in DMSO (final concentration of DMSO did not exceed 1%) and incubated for 48 h at 37 C, under 5% CO2 atmosphere. Cell growth monitoring was determined by measurements in microplates through a cellscreen® system. Cellscreen® consists of a microscope coupled to an image analyzer. A camera allows taking images of the surface of the wells of a microplate which is located on a motorized stand. The images are analyzed well by well by the processor and the surface of cell layer at the bottom of each well is measured. Development cell is evaluated by image analysis by determining the percentage of surface coated wells by cells. On the other hand, the potential cytotoxicity of molecules studied relative to Caco-2 cells in culture was determined using the neutral red or NRU (Neutral Red Uptake) that can adsorb on lysosomal membrane of viable cells. The neutral red test was performed after 48 h of incubation and the absorbance was measured at 540 nm. In case of death, the degraded lysosomal membrane does not allow the adsorption of neutral red. The absorbance at 540 nm is directly proportional to the amount of living cells (Mingoia et al., 2007). The number of live cells after treatment with selected

molecules to the number of untreated control concentration cells allows the evaluation of the cytotoxicity of these molecules. The results were expressed as IC50 mean values with the standard deviations. IC50 was defined as the concentration of a molecule leading to 50% cell mortality. The mortality rate (% cellular death) is calculated according to the following equation:

. 100 % cellular death ¼ 1 Absflavonolignans Absreference where Absreference is the absorbance of control without flavonolignans and Absflavonolignans is the absorbance obtained for the assay with cells treated with flavonolignans). 2.7. Statistical analysis All statistical analyses were performed using the software SPSS Version 22 (IBM® SPSS® Statistics, USA). Significant differences between the results were calculated by multiple sample comparison of the means (ANOVA) and the LSD test, with a significance level of p < 0.05. The error bars presented on the figures correspond to the standard deviations. 3. Results and discussion 3.1. Oil and fatty acids recovery In order to establish the SC-CO2 optimum conditions to recover oil from milk thistle seeds, different pressures (160e220 bars) and temperatures (40e80 C) were studied (Table 1). Two-way ANOVA analysis showed that pressure and extraction temperature had a significant influence (p < 0.05) on the amount of recovered oil (Fig. 3). As can be expected, when temperature was 40 C, oil recovery increased when pressure was augmented, observing the maximum oil recovery (31.83 ± 0.84%) when SC-CO2

Fig. 3. Oil recovery from milk thistle seeds using SC-CO2 process (160e220 bars, 40e80 C).


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Table 2 Fatty acids recovery from milk thistle seeds using supercritical carbon dioxide (SCeCO2) process (160e220 bars, 40e80 C). Pressure (bars)

Temperature ( C)

C16:0

160

40 60 80 40 60 80 40 60 80

8.74 10.04 9.62 9.12 7.44 8.72 11.23 6.46 10.89

180

220

C18:0 ± ± ± ± ± ± ± ± ±

1.07abc 2.00ab 0.46ab 0.64abc 3.00bc 0.82abc 0.56b 3.00c 1.32b

8.31 7.28 6.15 9.65 5.20 5.52 6.19 9.43 8.11

C18:1

± ± ± ± ± ± ± ± ±

1.97ab 2.53ab 0.68ab 4.71b 0.42a 0.59a 1.23ab 3.00b 1.32ab

22.56 19.68 22.59 29.22 20.17 21.05 21.61 19.77 24.83

C18:2 ± ± ± ± ± ± ± ± ±

3.78ab 2.11a 0.65ab 4.71c 0.42a 1.01ab 1.68ab 3.00a 1.32b

53.32 59.94 47.64 55.18 54.53 53.54 57.47 66.70 51.65

C20:0 ± ± ± ± ± ± ± ± ±

8.59ab 4.71ac 2.38b 9.95ab 3.00ab 5.42ab 3.20ac 4.77c 1.60ab

2.41 3.15 2.87 3.08 2.31 1.82 2.33 2.83 2.17

± ± ± ± ± ± ± ± ±

0.01a 0.71a 0.80a 0.21a 1.51a 0.55a 0.56a 0.48a 0.31a

aec

Different letters in the same column indicate significant statistical differences (p < 0.05).

Table 3 Flavonolignans recovery from milk thistle seeds using supercritical carbon dioxide (SCeCO2) process (160e220 bars, 40e80 C). Pressure (bars) 160

180

220

Temperature ( C) 40 60 80 40 60 80 40 60 80

SCN (mg/ml of oil) 46.79 40.36 48.31 66.45 57.34 52.26 72.93 58.38 60.80

± ± ± ± ± ± ± ± ±

ab

5.63 1.58a 10.23abc 3.00de 8.57cdf 8.15bc 4.71e 2.99cdf 1.32df

SDN (mg/ml of oil) 107.03 96.42 105.38 113.33 107.14 94.29 125.42 100.89 101.08

± ± ± ± ± ± ± ± ±

ab

4.95 4.70c 6.20ab 3.00a 6.82ab 3.13c 4.71d 4.46bc 1.32bc

SBN (mg/ml of oil) 96.47 105.28 70.20 129.26 105.88 74.88 140.24 131.49 51.10

± ± ± ± ± ± ± ± ±

a

4.80 5.73a 26.76b 3.00c 3.00a 6.43b 4.99c 5.15c 1.32d

SM (mg/ml of oil) 253.63 242.06 223.90 309.03 270.36 221.43 338.59 290.76 212.98

± ± ± ± ± ± ± ± ±

6.01ab 2.23ac 4.11cd 9.01e 17.52bf 2.06cd 6.12g 9.46ef 3.96d

SCN: Silychristin. SDN: Silydianin. SBN: Silybinin. SM: Silymarin. aef Different letters in the same column indicate significant statistical differences (p < 0.05).

at 220 bars and 40 C was used. However, it should be noted that oil recovery was significantly lower when temperature was increased for all pressures, especially at 60 C. This fact can be explained by a decrease in density when temperature was increased (Table 1), thus decreasing the oil solubility. These results are in close agreement to those reported previously by Celik and Guru (2015) when they studied the impact of SC-CO2 on oil recovery from milk thistle in similar conditions to those evaluated in the present work. They found a significant decrease in oil yield when temperature was increased. Moreover, other authors also found a significant decrease in oil yield from nutmeg seeds (Machmudah et al., 2006) and hemp seeds (Da Porto et al., 2012) when temperature was increased from 40 C to 60 C after applying 150 and 300 bars, respectively. So, it can be concluded, that 40 C was the optimal temperature to recover oil

Fig. 4. HPLC analysis of flavonolignans from milk thistle oil seeds.

under SC-CO2 conditions. On the other hand, chromatographic analysis by GCeMS revealed the presence of 7 fatty acids: myristic acid (C14:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), and arachidonic (C20:0) in all the SC-CO2 extracted oils, being oleic and linoleic acids the predominant (Table 2). Milk thistle oil presented an important content (70e85%) of unsaturated fatty acids. These results are in close agreement to those reported by Yin et al. (1998), who found values of unsaturated fatty acids between 69 and 75% of total fatty acids and those found by Li et al. (2012), who reported that unsaturated fatty acids were around 74% in milk thistle oil seeds. As can be seen in Table 2, linoleic acid is the most abundant acid (47.64e66.70%) extracted in milk thistle seeds using SC-CO2. It should be noted the importance of recovering this polyunsaturated fatty acid as it is an essential fatty acid, which forms part of the omega-3 family and has been involved in several physiological functions (Denisa et al., 2013). Oleic acid is also present at significant levels up to 19.68e24.83% of SC-CO2 extracted oils. This fatty acid is also of a high importance as can be used for both food and pharmaceutical industries. In this line, essential fatty acids can provide nutritional and dietary values and can justify its use for cardiovascular and drought and physiological aging of the skin diseases (Calani et al., 2012). Moreover, the results obtained in the present work showed that the extraction temperature had no effect on the levels of SC-CO2 extractable fatty acids. 3.1.1. Flavonolignans recovery On the other hand, the optimum conditions to recover flavonolignans from milk thistle under SC-CO2 extraction, were evaluated. After applying SC-CO2, the HPLC analyses identified and quantified the flavanolignan complex of silymarin (SM) (silychristine (SCN), silydianin (SDN) and silybin (SBN)) (Table 3, Fig. 4.). HPLC analysis showed that silychrisitin and silydianin levels obtained in the present study are significantly higher (with

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It is well established that supercritical fluid temperature to extract thermolabile compounds must be fixed between 35 C and 60 C; and near the critical point as low as possible to avoid degradation. The increased temperature reduces SC-CO2 density (for a fixed pressure) thereby reducing SC-CO2 solvent power; but it increases vapor pressure of extracted compounds. The results obtained in the present study were in close agreement to those obtained by Celik and Guru (2015), when they evaluated the effects of SC-CO2 (160e220 bars, 40e80 C) on the recovery of silybin from milk thistle seeds. They also found a significant decrease in the recovered amounts of silybin A and total silybin when temperature was augmented up to 80 C compared to lower temperatures. They attributed this phenomenon to a decrease in CO2 density, thus difficulting the extraction of silybin. Fig. 5. Antioxidant activity recovery from milk thistle seeds after applying supercritical carbon dioxide (SCeCO2) with co-solvent (160e220 bars, 40e80 C).

32.31 ± 2.37 mg/g of seeds (72.93 ± 4.71 mg/ml oil) and 39.32 ± 1.31 mg/g of seeds (125.42 ± 4.71 mg/ml oil), respectively) than those already reported by Quaglia et al. (1999) (0.47e6.9 mg/g of seeds) and Subramaniam et al. (2008) (3.12 mg/g of seeds). Silybin is the most active component of flavonolignans and its quantification is a key step for the determination of biological activity. Silybin is present at very high levels (40.98 ± 1.46 mg/g of seeds (140.24 ± 4.99 mg/ml oil)) and is higher than those of Quaglia et al. (1999) (1.43 mg/g of seeds) and Subramaniam et al. (2008) (23.15 mg/g of seeds). This difference may be attributed to flavonolignan's extraction method, pretreatment of seeds and seeds origin (soil, climate, etc.). As can be seen in Table 3, SBN and SDN were the predominant flavonolignans extracted by SC-CO2 while the amount of SCN was significantly lower. These results differ from those obtained from Quaglia et al. (1999) who found that SBN was the predominant flavonolignan found in milk thistle seeds followed by SDN and SCN. These differences can be attributed to the extraction technique and the origin of the plants used. In this line, other studies led to general conclusions about the parameters according to the plant matrix (oleoresins, essential, volatile and seed oils). The choice of extraction conditions depends on the specific compound. Thus the molecular weight and polarity should be taken into consideration (Reverchon and De Marco, 2006). Moreover, two-way ANOVA analysis was established to determine the effect of pressure and temperature on flavonolignans recovery from milk thistle under SC-CO2. The results showed that flavonolignans concentrations after SC-CO2 are greatly influenced by the conditions of pressure and temperature, although the behaviour varied according to the extracted flavonolignan (Table 3).

Fig. 6. Caco-2 cells specific growth rates (m, h1) in presence of flavonolignans and by supercritical CO2 extract (SBN: Silybinin, SCN: Silychristin, SDN: Silydianin). E: Extract obtained under SC-CO2 optimum conditions (220 bars, 40 C).

3.2. Antioxidant activity Milk thistle oil had significant scavenging effects on the DPPH radical (data not shown). This antioxidant activity reflects the bioactive molecules composition on oil extracted from S. marianum (1.1 ± 0.6% of polyphenols on dried material, 13.4 mg b-carotene/ 100 g of carotenoids and 70e85%% of unsaturated fatty acids). However, the ANOVA analysis did not show any significant differences in DPPH values of the SC-CO2 oil extracts when pressure and temperature were increased, thus obtaining DPPH values in the same order of scavenging reactivity (0.57 ± 0.12 mg/ml). On the other hand, as can be observed in Fig. 5, the ABTS values were significantly higher when SC-CO2 treatments at 220 bars (40 C and 60 C) were used compared to SC-CO2 at 160 and 180 bars. Moreover, the values at ABTS obtained after applying SCCO2 at 220 bars and 80 C were also lower than those obtained for the same pressure conditions at lower temperatures. This fact can be attributed to the degradation of some thermolabile and easily oxidable compounds (i.e vitamin C, vitamin E) when high temperatures and pressures conditions are combined. 3.3. Evaluation of cytotoxic activity Moreover, the anti-proliferative activities of the individual flavonolignans and the extracts obtained by SC-CO2 at the optimum conditions (220 bars, 40 C) against Caco-2 cancer cells, were evaluated (Fig. 6). Results showed that silychristin and silydianin did not exert a significant increase in antiproliferative properties when their concentrations were increased. However, silybin and milk thistle oil

Fig. 7. Mortality percentage of Caco-2 cells after 48 h of exposure with different concentrations of flavonolignans determined by the neutral red test (SBN: Silybinin, SCN: Silychristin, SDN: Silydianin). E: Extract obtained under SC-CO2 optimum conditions (220 bars, 40 C).


seeds extract obtained after applying SC-CO2 at optimum conditions showed a significant decrease in the proliferative activities in the concentration range from 50 to 100 mg/ml. Silybin and SC-CO2 extract have cytostatic properties but silychristin and silydianin did not exhibit any of these properties at the concentrations studied. In this line, Hogan et al. (2007) also reported that silybin is the most active flavonolignan of silymarin. Moreover, in the present work, the effects of the individual flavonolignans and the SC-CO2 extract obtained at 220 bar and 40 C and containing 32 mg/ml oil of silychristin, 39 mg/ml oil of silydianin, and 46 mg/ml (oil) silybin on Caco-2 cancer cells growth was observed after 48 h. In order to evaluate the cytotoxicity, the percentage of mortality percentage was studied using neutral red test. Fig. 7 presents the percentage of mortality of Caco-2 cancer cells for different concentrations of flavonolignans standards and SC-CO2 extract (E). It was found that silybin, silychristin and SC-CO2 extract induced a significant cell growth inhibition, thus leading to an arrest of cell growth at different concentrations of silybin (10, 20, 50 and 100 mg/ ml), silychristin (50 and 100 mg/ml) and SC-CO2 extract (50 and 100 mg/ml) (Fig. 7). The apparent growth rate of the cells in the presence of silychristin and silydianin was substantially similar to the control cells showing that these two flavonolignans had no significant effect on cells with mortality rates below 20%. In terms of silybinin, which corresponds to the most active flavonolignan, the cytotoxic effect at 50 and 100 mg/ml was significant with 42% mortality and 52% mortality, respectively. The extract obtained under SC-CO2 optimum conditions (220 bars, 40 C) also gave reasonable recoveries of flavonolignans and inhibited the proliferation of Caco-2 carcinoma cells in a dose-responsive manner. As it is shown in Fig. 7, SC-CO2 extract induced an important percentage of mortality (43e71 % for concentrations from 10 to 100 mg/ml). This can be due to the content of flavonolignans in the extract (silychristin 68 mg/ml, silydianin 121 mg/ml, and silybin 143 mg/ml). Cell growth inhibition when SC-CO2 extract was used at low concentrations can be explained by the fact that this extract is a mixture of four flavonolignans, and perhaps a synergistic effect can occur. Moreover, the existence of other active molecules in the extract could explain the observed effects. The purpose of this evaluation was to investigate the biological activity of the extract obtained from milk thistle after applying SC-CO2 extraction process with co-solvent at optimum conditions, which can have important benefits from a healthy point of view. Moreover, the IC50 values were calculated. It was found that oil seed extract exerted stronger cytotoxicity than silibinin since the IC50 values of cytotoxicity on Caco-2 were 96 mg/ml and 70 mg/ml, for silibinin and oil seed extract, respectively. Thus, although silibinin showed its potential cytotoxicity, some other active compounds may be responsible of the cytotoxicity of SC-CO2 extracts. Our results were consistent with those obtained by Hogan et al. (2007), which presented a cytotoxic effect of silybin on other colon cancer cell lines Fet, Geo, and HCT116. Some other studies also evaluated the cytotoxicity of silymarin towards different cancer cells and determined the most effective concentration. In this line, in a study performed on human epidermal A431 cells, it was demonstrated that silymarin cytotoxicity varied according to the concentration and exposure time (Ahmad et al., 1998). In addition, the antiproliferative effect of silymarin on breast cancer cells MDAMB468 was demonstrated (Zi et al., 1998). 4. Conclusions SC-CO2 with co-solvent technique is a useful tool to recover oil, fatty acids and flavonolignans from milk thistle seeds. This

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technique allowed the recovery of 7 fatty acids, being linoleic, oleic and palmitic acids the predominant fatty acids identified in S. marianum oil seeds. Moreover, four major flavonolignans (silychristin, silydianin, silybin and taxifolin) were recovered in the oil extracts from milk thistle obtained under SC-CO2 extraction. Pressure and temperature had a significant influence in the recovery of the valuable compounds, reaching the maximum recovery of oil and flavonolignans when SC-CO2 at 220 bars and 40 C was used. Oil extracts obtained after SC-CO2 under the optimum conditions presented an important antioxidant capacity. In addition, silybin and SC-CO2 extracts showed important cytostatic properties on Caco-2 cancer cell lines compared to silychristin and silydianin. The results obtained in the present study are of a great interest in the evaluation of the antioxidant and cytotoxic activities of flavonolignans extracted by SC-CO2. Further experiments can be considered towards other cancer cell lines to study the ability of flavonolignans extracted by SC-CO2 to inhibit their proliferation. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.07.006. References Ahmad, N., Gali, H., Javed, S., Agarwal, R., 1998. Skin cancer chemopreventive effects of a flavonoid antioxidant silymarin are mediated via impairment of receptor tyrosine kinase signaling and perturbation in cell cycle progression. Biochem. Biophys. Res. Commun. 247, 294e301. Barba, F.J., Grimi, N., Vorobiev, E., 2014. New approaches for the use of nonconventional cell disruption technologies to extract potential food additives and nutraceuticals from microalgae. Food Eng. Rev. 7, 45e62. Bearth, A., Cousin, M.-E., Siegrist, M., 2014. The consumer's perception of artificial food additives: influences on acceptance, risk and benefit perceptions. Food Qual. Prefer 38, 14e23. Bensebia, O., Barth, D., Bensebia, B., Dahmani, A., 2009. Supercritical CO2 extraction of rosemary: effect of extraction parameters and modelling. J. Supercrit. Fluids 49, 161e166. Bosch-Barrera, J., Menendez, J.A., 2015. Silibinin and STAT3: a natural way of targeting transcription factors for cancer therapy. Cancer Treat. Rev. http:// dx.doi.org/10.1016/j.ctrv.2015.04.008. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 28, 25e30. Calani, L., Brighenti, F., Bruni, R., Del Rio, D., 2012. Absorption and metabolism of milk thistle flavanolignans in humans. Phytomedicine 20, 40e46. Celik, H.T., Guru, M., 2015. Extraction of oil and silybin compounds from milk thistle seeds using supercritical carbon dioxide. J. Supercrit. Fluids 100, 105e109. Chemat, F., Vian, M.A., Cravotto, G., 2012. Green extraction of natural products: concept and principles. Int. J. Mol. Sci. 13 (7), 8615e8627. Chu, S.-C., Chiou, H.-L., Chen, P.-H., Yang, S.-F., Hsieh, Y.-S., 2004. Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol. Carcinog. 40, 143e149. Da Porto, C., Voinovich, D., Decorti, D., Natolino, A., 2012. Response surface optimization of hemp seed (Cannabis sativa L.) oil yield and oxidation stability by supercritical carbon dioxide extraction. J. Supercrit. Fluids 68, 45e51. Deng, Q., Zinoviadou, K.G., Galanakis, C.M., Orlien, V., Grimi, N., Vorobiev, E., Lebovka, N., Barba, F.J., 2014. The effects of conventional and non-conventional processing on glucosinolates and its derived forms, isothiocyanates: extraction, degradation, and applications. Food Eng. Rev. 1e25. http://dx.doi.org/10.1007/ s12393-014-9104-9. Denisa, I., Potierb, B., Vancassela, S., Heberdena, C., Lavialle, M., 2013. Omega-3 fatty acids and brain resistance to ageing and stress: body of evidence and possible mechanisms. Ageing Res. Rev. 12, 579e594. Downham, A., Collins, P., 2000. Colouring our foods in the last and next millennium. Int. J. Food Sci. Technol. 35, 5e22. El-Mallah, M.H., El-Shami, S.M., Hassanein, M.M., 2003. Detailed studies on some lipids of Silybum marianum (L.) seed oil. Grasas Aceites 54, 397e402. Eo, H.J., Park, G.H., Song, H.M., Lee, J.W., Kim, M.K., Lee, M.H., Lee, J.R., Koo, J.S., Jeong, J.B., 2015. Silymarin induces cyclin D1 proteasomal degradation via its phosphorylation of threonine-286 in human colorectal cancer cells. Int. Immunopharmacol. 24, 1e6. Fan, L., Ma, Y., Liu, Y., Zheng, D., Huang, G., 2014. Silymarin induces cell cycle arrest and apoptosis in ovarian cancer cells. Eur. J. Pharmacol. 743, 79e88. Friedrich, J.P., Pryde, E.H., 1984. Supercritical CO2 extraction of lipid-bearing materials and characterization of the products. J. Am. Oil Chem. Soc. 61, 223e228. Goodarznia, I., Eikani, M.H., 1998. Supercritical carbon dioxide extraction of

282


essential oils: modeling and simulation. Chem. Eng. Sci. 53, 1387e1395. Hadolin, M., Skerget, M., Knez, Z., Bauman, D., 2001. High pressure extraction of vitamin E-rich oil from Silybum marianum. Food Chem. 74, 355e364. Hogan, F.S., Krishnegowda, N.K., Mikhailova, M., Kahlenberg, M.S., 2007. Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer. J. Surg. Res. 143, 58e65. Jiang, K., Wang, W., Jin, X., Wang, Z., Ji, Z., Meng, G., 2015. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells. Oncol. Rep. 33, 2711e2718. Li, F., Yang, L., Zhao, T., Zhao, J., Zou, Y., Zou, Y., Wu, X., 2012. Optimization of enzymatic pretreatment for n-hexane extraction of oil from Silybum marianum seeds using response surface methodology. Food Bioprod. Process 90, 87e94. Machmudah, S., Sulaswatty, A., Sasaki, M., Goto, M., Hirose, T., 2006. Supercritical CO2 extraction of nutmeg oil: experiments and modeling. J. Supercrit. Fluids 39, 30e39. Mastron, J.K., Siveen, K.S., Sethi, G., Bishayee, A., 2015. Silymarin and hepatocellular carcinoma: a systematic, comprehensive, and critical review. Anticancer. Drugs. http://dx.doi.org/10.1097/CAD.0000000000000211. Mingoia, R.T., Nabb, D.L., Yang, C.-H., Han, X., 2007. Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome {P450} enzyme activity and inducibility, and its application in in vitro cytotoxicity screening. Toxicol. Vitr. 21, 165e173. NIST, 1986. National Bureau of Standards. National Institute of Standards and Technologies (NIST), United States of America. Parniakov, O., Barba, F.J., Grimi, N., Marchal, L., Jubeau, S., Lebovka, N., Vorobiev, E., 2014. Pulsed electric field assisted extraction of nutritionally valuable compounds from microalgae Nannochloropsis spp. using the binary mixture of organic solvents and water. Innov. Food Sci. Emerg. Technol. 27, 79e85. Pereira, C., Barros, L., Carvalho, A.M., Santos-Buelga, C., Ferreira, I.C.F.R., 2015. Infusions of artichoke and milk thistle represent a good source of phenolic acids and flavonoids. Food Funct. 6, 56e62. Quaglia, M.G., Bossu, E., Donati, E., Mazzanti, G., Brandt, A., 1999. Determination of silymarine in the extract from the dried Silybum marianum fruits by high performance liquid chromatography and capillary electrophoresis. J. Pharm. Biomed. Anal. 19, 435e442. Rawson, A., Tiwari, B.K., Brunton, N., Brennan, C., Cullen, P.J., O'Donnell, C.P., 2012. Application of supercritical carbon dioxide to fruit and vegetables: extraction, processing, and preservation. Food Rev. Int. 28, 253e276. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization

assay. Free Radic. Biol. Med. 26, 1231e1237. Reverchon, E., De Marco, I., 2006. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit. Fluids 38, 146e166. Reverchon, E., Senatore, F., 1992. Isolation of rosemary oil: comparison between hydrodistillation and supercritical CO2 extraction. Flavour Fragr. J. 7, 227e230. Rosello-Soto, E., Galanakis, C.M., Brncic, M., Orlien, V., Trujillo, F.J., Mawson, R., Knoerzer, K., Tiwari, B.K., Barba, F.J., 2015. Clean recovery of antioxidant compounds from plant foods, by-products and algae assisted by ultrasounds processing. Modeling approaches to optimize processing conditions. Trends Food Sci. Technol. 42, 134e149. Senorans, F.J., Ibanez, E., Cavero, S., Tabera, J., Reglero, G., 2000. Liquid chromatographic-mass spectrometric analysis of supercritical-fluid extracts of rosemary plants. J. Chromatogr. A 870, 491e499. Sharma, G., Singh, R.P., Chan, D.C.F., Agarwal, R., 2003. Silibinin induces growth inhibition and apoptotic cell death in human lung carcinoma cells. Anticancer Res. 23, 2649e2655. Subramaniam, S., Vaughn, K., Carrier, D.J., Clausen, E.C., 2008. Pretreatment of milk thistle seed to increase the silymarin yield: an alternative to petroleum ether defatting. Bioresour. Technol. 99, 2501e2506. Thelen, P., Jarry, H., Ringert, R.-H., Wuttke, W., 2004. Silibinin down-regulates prostate epithelium-derived ets transcription factor in LNCaP prostate cancer cells. Planta Med. 70, 397e400. Thurmond, T.S., 2014. U. S. Food and Drug Administration's (FDA) safety assessment of food ingredients. In: ACS Symp. Ser, vol. 1162, pp. 91e95. Tyagi, A.K., Agarwal, C., Chan, D.C., Agarwal, R., 2004. Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB468 cells. Oncol. Rep. 11, 493e499. Varghese, L., Agarwal, C., Tyagi, A., Singh, R.P., Agarwal, R., 2005. Silibinin efficacy against human hepatocellular carcinoma. Clin. Cancer Res. 11, 8441e8448. Wallace, S., Carrier, D.J., Beitle, R.R., Clausen, E.C., Griffis, C.L., 2003. HPLC-UV and LCMS-MS characterization of silymarin in milk thistle seeds and corresponding products. J. Nutraceut. Funct. Med. Foods 4, 37e48. Yin, Q., Wang, S., Nan, J., L.S, 1998. The fatty acid compositions of Silybum marianum by GC-MS. J. Yanbian Univ. Sci. 24, 26e28. Zi, X., Feyes, D.K., Agarwal, R., 1998. Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin dependent kinases and associated cyclins. Clin. Cancer Res. 4, 1055e1064.